PROJECT SUMMARY Mesenchymal stem cells (MSC) represent a promising cell source for diverse regenerative medicine applications. Isolated MSC retain their self-renewal capacity, have the potential to differentiate into multiple lineages, are hypoimmunogenic, and can home to injured tissues. In the context of musculoskeletal applications, transplanted MSC enhance bone, cartilage, and intervertebral disc repair in pre-clinical models and initial clinical trials. MSC secrete a myriad of cytokines, growth factors and metabolites that modulate immune responses and promote regenerative activities. However, the extremely low survival and engraftment of transplanted MSC significantly limit these cell-based therapies. A major hurdle to MSC survival, engraftment, and function is the lack of appropriate biomaterial delivery vehicles. During the current funding period, we engineered integrin-specific synthetic hydrogels that modulate MSC survival, engraftment, immunomodulatory secretome, and reparative activities in non-healing bone defects. Because of the use of human cells, these studies were limited to immunodeficient mice. The objective of this renewal application is to engineer synthetic hydrogels that promote MSC survival, immunomodulatory properties, and bone repair in more relevant immunocompetent models. Our central hypothesis is that integrin-specific hydrogels will support MSC survival and immunomodulatory properties to direct host immune cell-dependent actions to enhance bone repair. The scientific premise for this project is based on our compelling results with engineered integrin-specific materials that enhance transplanted MSC survival, functions, and bone repair and strategies to harness pro-healing monocyte populations. The rationale for this research is that it will establish bioactive cell delivery vehicles that enhance MSC survival and immunomodulatory and reparative functions. Aim 1: Engineer synthetic hydrogels that promote MSC immunomodulatory secretome and activities. Aim 2: Evaluate the ability of engineered hydrogels to support MSC survival and immunomodulatory properties to direct host immune cell-dependent actions for enhanced bone repair. The proposed research is highly innovative because it focuses on engineering synthetic hydrogels to control MSC survival, immunomodulatory properties, and bone repair. The use of humanized mice is also novel and will provide critical insights on hMSC-based therapies. This research is expected to yield the following significant outcomes. First, we will engineer synthetic hydrogels that promote MSC survival and immunomodulatory secretory and reparative activities. Second, we will establish the extent to which MSC direct host immune cells in bone repair. Finally, this research will provide direct comparisons for MSC survival and function across models of increased immune complexity. Because of the transformative potential of MSC, this research will have broad significance and impact to many regenerative medicine applications.